1. Field of the Invention
The present invention relates to methods for quantitative analysis for gene expression using as an analysis sample a library of single-cell derived cDNA fixed onto a carrier, which is formed by immobilizing cDNA derived from a single-cell onto a carrier. The present invention also relates to methods for quantitative analysis for gene expression using as an analysis sample a library of cDNA fixed onto a carrier, which is formed by immobilizing cDNA derived from a small number of cells onto a carrier.
2. Background Art
As conventional methods for gene expression analysis, typically mention is made of a microarray method and a real-time PCR method. In these conventional methods, a tissue slice consisting of a plurality of cells or a plurality of cultured cells (about 106) is often used as an analysis sample. In actual cells, since the expression amount of a gene variously changes with time, the expression of the gene varies per cell with time depending upon the site even in the same tissue. Therefore, when tissues and a plurality of cells are analyzed as a sample, variances between cells in measurement value are usually averaged. From the data thus obtained, it is difficult to obtain detailed information of a living organism having dynamical changes. Now that individual cells, which are minimum units of life for carrying out a biological activity, have different life phenomena from each other, it is difficult to integrally analyze living activity in a single cell and between cells by conventional gene expression analysis using a plurality of cells as an analysis sample.
Recently, there has been a growing trend to see life as a system. A minimum unit of the system of life is a cell. To elucidate the system of life, it is necessary to analyze all molecules contained in a single-cell and elucidate the cell integrally. For example, in the tissues concerning with in-vivo information transmission involving hormones and neurotransmitters and the embryo tissue in a development stage, it is considered that the amount of gene expression greatly differs between cells. Therefore, gene expression analysis at a single-cell level is highly required.
Recently, with remarkable technical developments in reagents and detection devices, etc., analysis of gene expression at a single-cell level has become feasible and interesting findings in the molecular biology have been reported and received attention. However, the amount of mRNA contained in a single-cell is extremely low. A problem of detection sensitivity has not yet completely solved and a gene that can be analyzed is still limited to highly expressing ones.
At the era of genome analysis, technical development has been made with focusing on how efficiently an unknown DNA sequence is read. In contrast, at the post genome era, to interpret a cell as a system, it is required to develop a means for analyzing, with a high sensitivity, from which gene mRNA expressed in individual single-cells is derived, and how much amount the mRNA is present. Under the circumstances, to efficiently recover mRNA molecules and synthesize cDNA, a method of capturing mRNA by use of oligo (dT) fixed magnetic beads and subjecting the mRNA to reverse transcription has been proposed (JP Patent Publication (Kokai) Nos. 2002-238575 and 2005-46138).
The aforementioned conventional techniques may have the following technical problems. First, in the methods according to these patent documents, 1 μg of RNA (corresponding to the amount of RNA of 105 cells) is extracted and purified from a plurality of cells in advance and diluted to prepare an RNA solution containing a small amount of RNA, which is used as a starting material. More specifically, RNA, in actual, is not extracted from a single-cell. The RNA extraction methods and purification methods set forth in the patent documents require operations of repeatedly transferring a sample from a tube to another tube, allowing a column to absorb RNA and repeatedly washing the column. Through these operations, a sample of RNA runs out. Therefore, it is difficult to recover an extremely small amount of RNA (about 10 pg) contained in a single-cell. Furthermore, in such conventional methods, recovering rates differ between individual samples. Thus, they are unfavorable for quantitative analysis. Furthermore, in each of the methods according to the patent documents, after reverse transcription is performed on the magnetic beads, a customary PCR amplification is performed. The absolute total number and ratio of RNA molecules derived from various types of genes which are present in an original sample are varied by the amplification step. Therefore, these mRNA cannot be used as a sample for quantitative analysis.
On the other hand, a reagent kit for a single-cell gene expression analysis is commercially available, by which a cell lysis step, DNase treatment step, and reverse transcription step are carried out in a single tube. Synthesis of cDNA by the reagent kit is easily performed and free from conventional problems such as a loss of an analysis sample during extraction and purification processes of a nucleic acid and variance in yield. However, a residual reagent remains in a cDNA solution synthesized by the reagent kit and inhibits a PCR amplification reaction. Therefore, it is difficult to perform a real-time PCR analysis using the whole amount of cDNA derived from a single-cell. To reduce the inhibition of PCR amplification by the residual reagent, cDNA derived from a single-cell is further divided into portions and a portion has to be used for analysis. As a result, detection sensitivity of the real-time PCR analysis greatly decreases. In addition, cDNA derived from a single-cell is consumed as analysis proceeds. The types of genes that can be analyzed are limited.